Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.

Vibe-Pedersen, Karen; Kornblihtt, Alberto R.; Baralle, Francisco E.

doi:10.1002/j.1460-2075.1984.tb02165.x

Cited by 96 publications

(58 citation statements)

References 35 publications

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“…We report here that the type III repeats are indeed represented by a repeating structure in the gene, comprising two exons, which together encode a complete repeat. Furthermore, analyses of the gene structure presented here and elsewhere (27,29) show that variation in the number of type III repeats arises by a form of alternative splicing in which the sequences encoding an entire repeat can be included or omitted. …”

mentioning

confidence: 69%

“…Secondary structure predictions suggest that the repeating structure of the protein is further subdivided into smaller structural units and that these also correspond with exons in the gene. (23)(24)(25)(26)(27)(28)(29)). An interesting problem posed by these results is the way in which the various functional domains and repeating structural units that make up a fibronectin subunit are encoded in the exons of the gene.…”

Section: Introductionmentioning

confidence: 99%

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Repeating modular structure of the fibronectin gene: relationship to protein structure and subunit variation.

Odermatt¹,

Tamkun²,

Hynes³

1985

Proc. Natl. Acad. Sci. U.S.A.

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Analysis of the exon-intron structure of the rat fibronectin gene shows that exons correspond precisely with repeating structural units in the protein and that alternative use of some exons produces fibronectin subunits that differ by the presence or absence of certain structural modules. Secondary structure predictions suggest that the repeating structure of the protein is further subdivided into smaller structural units and that these also correspond with exons in the gene. (23)(24)(25)(26)(27)(28)(29)). An interesting problem posed by these results is the way in which the various functional domains and repeating structural units that make up a fibronectin subunit are encoded in the exons of the gene. We report here that the type III repeats are indeed represented by a repeating structure in the gene, comprising two exons, which together encode a complete repeat. Furthermore, analyses of the gene structure presented here and elsewhere (27,29) show that variation in the number of type III repeats arises by a form of alternative splicing in which the sequences encoding an entire repeat can be included or omitted. MATERIALS AND METHODSEnzymes were purchased from New England Biolabs, except for the calf alkaline phosphatase (Boehringer Mannheim).

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mentioning

confidence: 69%

Section: Introductionmentioning

confidence: 99%

Repeating modular structure of the fibronectin gene: relationship to protein structure and subunit variation.

Odermatt¹,

Tamkun²,

Hynes³

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…The mechanism of this increase is unknown. Measurement (42,(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57). Whereas such variations appear to relate, at least in part, to cell specificities producing the fibronectin (48,53), it is not known if the process of alternative splicing relates in any fashion to modulating fibronectin mRNA levels for a given cell.…”

Section: Resultsmentioning

confidence: 99%

Modulation of fibronectin gene expression in human mononuclear phagocytes.

Yamauchi

Martinet²,

Crystal³

1987

J. Clin. Invest.

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Under some conditions, mononuclear phagocytes spontaneously synthesize and release fibronectin, an extracellular matrix glycoprotein with versatile effects on cell-matrix interactions. To gain insight into the processes that modulate the level of fibronectin secretion by these cells, we used monocytes, in vitro matured nonocytes and alveolar macrophages as models to compare fibronectin mRNA levels and fibronectin secretion in a variety of circumstances. Using Northern analysis and dot-blot analysis with a 32P-labeled human fibronectin cDNA probe, we evaluated steady-state mRNA levels and a human fibronectin-specific ELISA was used to evaluate fibronectin secretion. In all cases the amounts of fibronectin secreted paralleled fibronectin mRNA levels. Specifically (a) when fibronectin mRNA was undetectable, as in the case of normal blood monocytes, no fibronectin was secreted, but whenever fibronectin mRNA was present, as in normal alveolar macrophages, fibronectin was secreted by the cells; (b) as monocytes matured into macrophages in vitro, the cells began to express fibronectin mRNA and the cells secreted fibronectin; (c) when alveolar macrophages were activated with surface stimuli such as lipopolysaccharide (LPS) or immune complexes, fibronectin mRNA levels decreased and in parallel, the cells secreted less fibronectin; (d) in idiopathic pulmonary fibrosis (IPF), alveolar macrophages contained severalfold more fibronectin mRNA transcripts that normal and the cells spontaneously secreted severalfold more fibronectin than normal; and (e) when IPF alveolar macrophages were placed in culture the fibronectin mRNA levels in the cells decreased with time, and concurrently the amounts of fibronectin produced per unit time continually decreased. The observation of a strict concordance of fibronectin mRNA levels and fibronectin release by mononuclear phagocytes suggests that, at least in many circumstances, fibronectin secretion by mononuclear phagocytes is controlled by steady-state levels of fibronectin mRNA.

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“…In comparison with the minigene system widely used to study other splicing models (13)(14)(15)(16)(17), where only one exon and its flanking regions are cloned in a heterologous gene context, the apoA-II expression system that we generated contains most of the elements necessary for its transcription and RNA processing, which are also present in the endogenous apoA-II gene. Such a construct should allow the study of the cis-acting elements affecting apoA-II exon 3 splicing in a context as close as possible to the chromosomal background.…”

Section: Resultsmentioning

confidence: 99%

An Exonic Splicing Enhancer Offsets the Atypical GU-rich 3′ Splice Site of Human Apolipoprotein A-II Exon 3

Arrisi-Mercado

Romano

Muro

et al. 2004

Journal of Biological Chemistry

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Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3 splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3 splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3 splice site.Pre-mRNA splicing is the process by which introns are removed and exons are joined together by a two-step trans-esterification reaction carried out by the spliceosome, a dynamic 60 S ribonucleoprotein particle (1). Formation of the spliceosome at particular splice junctions is triggered by recognition of the 5Ј splice site by the U1 small nuclear ribonucleoprotein and of the 3Ј splice site by U2AF followed by the U2 small nuclear ribonucleoprotein recognition of the branch point (2).The polypyrimidine tract is one of the important cis-acting sequences present in the 3Ј splice site of introns. The progressive deletion of the polypyrimidine tract abolish lariat formation, spliceosome assembly, and consequently the splicing process (3, 4), whereas increasing the length of the pyrimidine run can lead to improved efficiency of splicing in some systems (4).In vitro studies have demonstrated that the ability of polypyrimidine tracts to favor specific 3Ј splice site selection is not only determined by its length but also by its composition (4, 5). This feature coexists with a degree of flexibility in the specific sequence of a given tract.The human apolipoprotein A-II (apoA-II) 1 gene presents a peculiar arrangement of GT repeats within the polypyrimidine tract region at the intron 2/exon 3 junction that devia...

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Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.

Cited by 96 publications

References 35 publications

Repeating modular structure of the fibronectin gene: relationship to protein structure and subunit variation.

Repeating modular structure of the fibronectin gene: relationship to protein structure and subunit variation.

Modulation of fibronectin gene expression in human mononuclear phagocytes.

An Exonic Splicing Enhancer Offsets the Atypical GU-rich 3′ Splice Site of Human Apolipoprotein A-II Exon 3

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